Chitosan is an abundant,biocompatible and biodegradable biomaterial,and its degradation products are harmless natural metabolites,and non-antigenic.Chitosan is an amino-polysaccharide that under moderately acidic conditions (pH < 6) is a soluble,cationic polyelectrolyte.At higher pH,chitosans amino groups are deprotonated and the polymer becomes insoluble.Due to its pH dependent solubility,porous structure,and dense amine groups,it forms stable films on various surfaces and well-suited for a matrix for the attachment of biomolecules,such as proteins,enzymes,antibodies,and DNA.This paper describes a light-addressable electrolytic system used to perform an electrodeposition of enzyme-entrapped chitosan membranes for multiplexed enzyme-based bioassay using a digital micromirror device (DMD).In this system,a patterned light illumination is projected onto a photoconductive substrate serving as a photo-cathode to electrolytically produce hydroxide ions,which can lead to an increased pH gradient.The high pH generated at the cathode can locally cause gelation of chitosan through sol-gel transition.By controlling the illumination pattern on the DMD,a light-addressable electrodeposition of chitosan membranes with different shapes and sizes,as well as multiplexed micropatterning,was performed.The effects of the chitosan concentration and applied current density on the dimensional resolution of chitosan membrane formation were experimentally examined.Moreover,multiplexed enzyme-based bioassay of enzyme-entrapped chitosan membranes was also successfully demonstrated through the electrodeposition of the chitosan membranes with various shapes/sizes and entrapping different enzymes.As a model experiment,glucose and ethanol were simultaneously detected using shape-coded chitosan membranes entrapped with glucose oxidase (GOX),peroxidase (POD),and Amplex Red (AmR) or alcohol oxidase (AOX),POD,and AMR by using same fluorescence indicator (AmR) in a single detection chamber without cross-talk.